The present invention relates to a method for the detection of analytes, particularly to analytes which are present in only small quantities, as well as to kits and reagents for use in the detection method.
Reactions are widely used in the assay, testing and measurement of chemical, biochemical and biological analytes (e.g. immunoassays). The reaction between an analyte and particular reagents is usually the basis of specificity of such assays. While chemical reagents are the basis for some assays, the higher affinity reaction between analytes and biological molecules (e.g. enzymes, antibodies, nucleic acids) introduces higher specificity and sensitivity into assay procedures. Those reactions providing the highest specificity and sensitivity assays often do not involve the covalent modification of the analyte (e.g. chemical or biochemical enzyme reaction), and instead rely on the binding of the analyte to the specific reagent (e.g. antibody). Many of these covalent and binding reactions are carried out in solution, so that the reaction product and the analyte are present in the same phase. Reactions are detected by various methods such as a change in the optical (e.g. a colour, fluorescent, luminescent, light scattering) or electrochemical (amperometric, potentiometric, conductimetric) properties of the assay or text mixture.
Recent improvements have included the immobilisation of assay reagents for example onto solid supports such as beads or other surfaces. This allows the signal generated to be localised in the same plane. It is therefore more concentrated at this position and so easier to detect. Furthermore, problems with background xe2x80x9cnoisexe2x80x9d signals generated in the bulk of the analyte can be minimised. Examples of such surfaces include lipid films or membranes. Visible signals may be detected in these systems, for example as described in WO 98/00714 and WO 96/25665, or alternatively changes in conductance as a result of the use of ion gating may give rise to analyte detection, for example as described in WO 90/08783 or WO 89/01159.
Liposomes have been used in immunoassays, both as labels to carry a payload of signal molecules, and to release signal molecules in proportion to the level of analyte in the assay medium. In one such method, (W. J. Litchfield et al., Clin. Chem. (1984) 30(9), 1441-1445) liposomes have been lysed to release a signalling means such as a fluorescent dye or enzyme, into an assay reaction mixture in order to generate a signal. The signal however, is diluted throughout the reaction mixture.
The applicants have found an improved process for detecting analytes.
Furthermore, it is known that their are a plurality of methods of varying the output signal parameters of a marker by means of alteration of the energy state of the marker by means of application of a force or field to it. In many instances the force/field may be applied by the locality of a second, or more, entities. An example known in the art is the self quenching of the calcein fluorophore at high concentrations. A second example is the quenching of calcein, and similar fluorescent dyes, by the addition of ions. It is known in the art that cobalt ions quench many fluorophore species. However, the inventors have discovered new effects associated with the use of cobalt ions and other chemicals, which assist in signal generation both in the method of the invention and more generally.
The present invention provides a process for detecting an analyte which process comprises (a) contacting a sample suspected of containing said analyte with a containment means comprising a barrier which separates signal generating reagents from said sample, in the presence of an element which interacts specifically with said analyte, under conditions whereby interaction between the analyte and the said element results in activation of the signal generating reagents within the containment means on the side of the barrier opposite to the sample, and (b) detecting any signal generated and retained within the containment means from the sample side of the barrier.
In this process, although the analyte is free to interact directly or indirectly with the containment means, the signal is generated and retained by the barrier within the containment means. This provides concentration of the signal which is therefore easier to detect.
There is the possibility of amplification of a signal within the containment means. Very small numbers of analyte molecules or even a single analyte molecule, can generate a detectable signal within the containment means, making the system very sensitive for the detection of analyte which is present in only very small quantities.
In one embodiment of the invention, the said element is present on the barrier surface of said containment means prior to interaction with said analyte. In general, interaction with the analyte will result in removal of this element, and this in turn allows transport through the surface of signal activation reagents.
Alternatively, the element may be added to the sample. In this case the element may be associated with a reagent which permeabilises or otherwise allows transport of signal activating reagents through the surface of the containment means such that it blocks or competes with the activity of that reagent. Interaction of the analyte with the element releases this reagent which is then free to react with the containment means so as to allow activation of the signal generating reagents.
In yet a further embodiment, activation of the signal generating reagents may be dependent upon the formation of a complex between the analyte and the element. The thus formed complex may activate the signal generating means for example by interacting with the barrier of the containment means so as to allow signal activation to occur.
The containment means suitably comprises a solid or semi-solid structure whereas the sample may comprise a solid, semi-solid, liquid or a gaseous reagent. Suitably the containment means is in a different physical phase to the sample.
The containment means is suitably of particulate form, in which no one dimension of the 3-dimensional shape of each particle is greater than 4 times any other dimension. In other words, the ratio of the x:y or y:z or x:z or vice versa is not greater than 1:4. Examples of such particles are broadly spherical, such as polymer beads like nanospheres, nanoparticles of microparticles or membrane structures such as vesicles and liposomes. In this case the signal generating agents are contained inside the particle. They are suitably introduced during processes for the production of the particulate containment means.
Particulate containment means can be produced using conventional methods. For example, polymer particles can be produced using a range of processes including the use of phase separation in mixed phase emulsion systems, aggregation and agglutination reactions, extrusion as polymerising or setting beads, and from aerosols. Liposomes and vesicles can be produced by various encapsulation technologies which are known in the art, such as sonication, extrusion and detergent dialysis.
A suitable liposome composition comprises for example a phosphatidylcholine, cholesterol and dihexadecyl phosphate as illustrated hereinafter although other liposome compositions will be apparent to the skilled person. It may be preferable for stability purposes, for the liposomes to be biotinylated in the sense that they incorporate a biotin reagent such as biotinoyl dipalmitoyl phosphatidylethanolamine (biotin-DPPE).
Alternatively, the containment means may comprise a discontinuity, such as a pore structure in a solid or colloid surface, which may be closed to form a complete barrier to sample. Suitable solids include ceramic materials such as glass, metal oxides or silicon. Discontinuities can be introduced into such materials by phase separation or etching processes, or by adding a pattern lithographically or by anodisation. The signal generating agents are suitably introduced into the thus formed discontinuity. The opening to the discontinuity is then closed by the application of a reactive closure means, such as a lipid membrane or layer, which acts as a barrier to prevent ingress of test sample. The reactive closure means can however interact directly or indirectly with any analyte present in a sample applied to it. As a result, signal is generated within the confines of the discontinuity and reactive closure means, which can be detected by observation of reactive closure means from the sample side.
In general, the smaller the size of the containment means, the fewer the number of analyte molecules which are required to interact with the surface of the containment means in order to generate a signal there within. Therefore, where high levels of sensitivity is required, the containment means is made as small as possible. However, the smaller the volume of the containment means, the less signal generation agents can be contained within them and so the detection of the signal or of the containment means itself can be more difficult.
Suitably the containment means of the invention is between 1 nm and 100 xcexcm across, for example of from 1 nm to 100 xcexcm diameter where the containment means is generally circular in cross section. For example, the containment means may be of from 10 to 500 nm in size. A particle of dimensions of about 100 nm in diameter provides in practice, a good compromise between transducing a few molecular detection events into a signal which is readily detectable by single particle detection technology. The volume within a 100 nm particle or discontinuity may be of the order of 10xe2x88x9218L. Volumes of this size may contain a plurality of protein molecules, for example up to 20 protein molecules, or a larger number, for example up to 105 small molecules such as fluorescent dyes.
In a preferred embodiment, the containment means comprises a lipid membrane through which transport of a signal activating means or reagent is inhibited for example as a result of the presence of specific binding elements such as antibodies or binding fragments thereof. Where the containment means comprises a liposome, the entire surface may comprise lipid membrane. Alternatively, the lipid membrane may encapsulate or bind another particle such as a nanosphere, nanoparticle or microparticle, or can be introduced across a discontinuity in a solid surface such as a pore in a ceramic material as outlined above, using conventional methods.
Lipid membranes can prevent significant transport of all molecules other than water or specific molecules which are known to disrupt the membrane structure. Therefore, when used in accordance with the method of the invention, the membranes achieve the selective or specific transport of molecules such as ions, analytes or enzyme substrates into the containment means in order to activate the signal generating reagents, but prevent the signalling agents leaving the containment means.
The precise molecules which can be used to achieve this effect will depend upon various factors such as the nature of the containment means and the size of the signal generating reagents employed. For instance, where the signal generating reagents comprise large enzymes, it is necessary to ensure that, on interaction with an analyte, the membrane is disrupted or permeabilized only to the extent necessary to allow an activating agent such as an enzyme substrate to pass through.
Thus in a preferred embodiment, the interaction between the analyte and the element results in the appearance of a transport mechanism through the surface of the containment means, typically by a perturbation or change in permeability of the surface such as the opening of a channel, so as to allow ingress or egress of moieties which cause activation of the signal generating reagents, but not egress of the signal from the containment means.
Suitably the said element is an antibody or a binding fragment thereof which specifically binds the analyte. Alternatively, it may comprise other ligand binding proteins such as receptors, lectins, avidin or streptavidin.
The nature of the moieties required in order to activate the signal generating means will depend upon the nature of the signal generating means themselves. However, they may be simply comprise ions as illustrated hereinafter.
Suitable surface active and detergent like molecules will be readily apparent to the skilled person. Examples include switched ion carriers, enzyme substrate channels or inserted catalysts or cofactors as illustrated hereinafter.
A range of carrier molecules, such as Crown ether or peptide structures are able either to carry ions across lipid membranes (e.g. valinomycin, monensin) or produce channels in membranes (e.g. alamethicin, gramicidin). However, when attached to a large typically hydrophilic molecule such as a protein or nucleic acid, the transport ability of the molecules is inhibited.
Use can be made of these properties. For example the analyte interaction may be linked to a binding reaction with the large molecule so that it competes with the carrier molecule for binding to the hydrophilic molecule, or displaces the hydrophilic molecule from the carrier molecule. The change in transport of ions across the membrane can be detected by measuring for example the effect of the ion on the transmembrane electrochemical gradient (membrane potential, pH gradient using reagents such as dyes which accumulate and/or change their optical properties, such as fluorescent membrane probes), the interaction of the ion with a chromophore contained in the signal generating reagents, or by the activation of an enzyme by the transported ion.
Another range of molecules, typically peptides, allow the selective permeabilization of membranes to small molecules such as enzyme substrates. A typical peptide of this type is the peptide xe2x80x9cmini-GALAxe2x80x9d of structure LAEALAEALEALAA (SEQ ID NO:1), xe2x80x9cGALAxe2x80x9d (SEQ ID NO:2) of structure WEAALAEALAEALAEHLAEALAEALEALAA (SEQ ID NO:3), xe2x80x9cKALAxe2x80x9d (SEQ ID NO:4) of structure WEAKLAKALAKALAKHLAKALAKALKACEA (SEQ ID NO:5) and myristryl-mini-GALA. Other examples include melittin peptides and derivatives and variants thereof which have membrane permeabilising properties, such as those illustrated hereinafter. These peptides permeabilise the membrane to small molecules while preventing passage of larger molecules such as enzyme signal generating reagents and the signal agents produced thereby. The activity of the peptides may be blocked in the presence of a sensing reagent such as an antibody. However, if the antibody is specific for a particular analyte, it may be removed by said analyte, thereby allowing the peptide to exert its permeabilising effect so as to allow passage of the signal activation agents for example the enzyme substrates to pass into the containment means. The complex of the peptide and the sensing agent may be present in the lipid layer so that removal of the sensing agent has a direct effect on the surface of the layer. Alternatively, the peptide-sensing agent complex may be present in the surrounding solution with the sensing agent blocking the efficacy of the peptide. Removal of the sensing agent by interaction of the analyte will therefore free the peptide and allow it to exert its permeabilising effect on a liposome into which it then comes in contact.
In a modification of this approach, use may be made of signalling peptides, such as (FITC)-MLSLRQSIRFFKPATRTLCSSRYLL (SEQ ID NO:6) (P25) and (FITC)-MLSLRQSIRFFKPSTRTLCSSRYLLQQKPVVKTA (SEQ ID NO:7)-amide (P34), which insert themselves into a membrane and appear at the contained side. This can be demonstrated using fluorescent probe methods. Such peptides can be used to transport signal activation agents such as enzyme co-factors, metal ion complexes with catalytic activity (e.g. porphyrins) or even large hydrophilic structures such as soluble enzymes across the membrane, once a sensing or blocking reagent has been removed by reaction with the analyte. The said blocking agent may be displaced by reaction with the analyte or the analyte may compete for binding with the blocking agent. In either case, the amount of transport across the surface of the particle is related, typically proportionally, to the analyte.
Chemically modified forms of the signal peptides (e.g., P25) can insert into liposome-contained protease (e.g., trypsin) to produce a contained reaction. Specific fluorogenic substrates for proteases are well known (e.g., umbelliferone fluorescence on cleavage of the peptide) or can be simply detected by co-containment of pH sensitive fluorophores. Such forms may be used in the present invention.
For example, the leader sequence P-25 can be synthesised with a new N terminal amino acid linked to the remaining peptide by an ester linkage. Insertion of this N terminal group, guided for example by a reagent such as cardiolipin, results in the hydrolysis of the ester and a change in the fluorescence of the particular liposmes with which the peptide interacts. Selection of the nature of the amino acid allows a variety of proteases specific for that amino acid to be used.
Suitable signal generating means include those which are well known to those in the art and may comprise any of the signal or label generating reagents known for example in the field of immunoassays. For example, signal may be produced as a result of an enzyme reaction, which may be catalysed by a cofactor.
Signal generating agents may comprise agents or reagents which are capable of generating a light signal such as fluorescent dyes or luminescent enzyme systems like the luciferin/luciferase system. Alternatively, any of the other enzymatic detection systems such as the alkaline phosphatase reaction can be used. An example of an non-enzyme catalysed signal reaction would be the detection of lipid peroxidation from organic peroxides, which is catalysed by porphryins.
Rather than containing active enzyme, nanospheres of inactive enzyme can be produced, whose reactivation can be triggered by peptides. Inactive enzyme can be produced by co-containment of inhibitors and enzyme. Most competitive and many non-competitive inhibition of enzymes can be reversed by removal or dilution of the inhibitor. It will be understood that the means described for triggering entry of substrate can also be used to allow inhibitors to escape the nanosphere. Many enzymes, including but not restricted to oxidoreductases, require co-factors in order to operate. Such co-factors may be introduced by peptides forming channels or pores (allowing ingress or egress of other similar molecules) or via their specific insertion when attached to the above peptides (not allowing transport of other similar molecules) Some co-factors are normally tightly associated with the enzyme. The removal of such co-factors produces apoenzyme. Liposome-contained apoenzyme co-factors required by enzymes can be coupled to such peptides so that they are reconstituted with the enzyme. A typical example is the removal of the flavine adenine dinucleotide (FAD) co-factor from glucose oxidase to produce apo-glucose oxidase (Methods in Enzymology Vol. 92 Part E pages 413-417). FAD cofactor can be coupled to such peptides (e.g. by carbodiimide coupling to N6 (2-aminoethyl)-FAD. Glucose oxidase can be incorporated into liposomes by the procedures described herein for alkaline phosphatase
Alternatively, catalytic activity may be transported into the containment means. For example, the biological function of signal peptides is to transport polypeptides and proteins through membranes. Indeed, the P25/34 signal peptide structure described above are responsible for inserting cytochrome oxidase through membranes. Such peptides can also be used to insert other non-enzymic catalytic activities through membranes. Inorganic or organic catalytic structures can be attached to peptides to introduce and contain the activity in nanospheres and liposomes. The reaction catalysed may require energy, which can be provided in the form of light absorbed by the catalytic centre (photo-catalysis) or in the form of heat, which may also be provided local to the contained reaction by absorption of light, other electromagnetic or ultrasonic radiation. The use of externally applied energy provides additional means of triggering the contained reaction. Other catalytic centres do not require energy. Radical reactions catalysed by haem are a typical example. For example, haem catalysed peroxidation of lipids can be achieved.
Haem groups catalyse a radical mediated chain reaction which leads to lipid peroxidation. Because of the chain reactions involved, very small concentrations of haem cause significant peroxidation. The lipid peroxidation has been conveniently demonstrated by monitoring the change in permeability of the lipid membrane using fluorescent dyes (as before), but the radicals could also be captured in other reactions.
The applicants have found a new signalling system which is particularly useful in the context of the present invention. While many dyes form coloured complexes with metal ions, few such complexes are fluorescent. Such dyes may not be retained following triggering of the reaction and their fluorescence may be self-quenched at useful concentrations. Further, metal ions are a well known means of quenching fluorescence.
It was found however, that interaction of certain dyes, such as ELF-97 (Molecular Probes Inc. USAxe2x80x94see xe2x80x9cHandbook of Fluorescent Probes and Research Chemicalsxe2x80x9d by Richard Haugland, 6th Ed. 1996) with cobalt ions induced fluorescence and shifted the fluorescence of the dye to the red end of the spectrum. This can be useful in the context of the present invention where interaction may be allowed as a result of the interaction of the analyte.
Furthermore, it was found that alkaline phosphatase may at certain pH values, act as a catalyst for certain dye substrate reactions, in particular the ELF-97 substrate reaction. Since this pH is of the same order as that at which the GALA peptide is active, this can form a preferred system for use in the invention.
Because, in accordance with the invention, signal generation takes place within an enclosed containment means, non-specific signal from the sample may be minimised by adding signal quencher reagents to the sample. Suitable quenching reagents will depend upon the nature of any signal generating means present in the sample. However, in a finding of the present inventors, para-nitrophenyl phosphate (PNP) a substrate used in a calorimetric assay, was found to quench signal from the fluorescent dye ELF-97.
In a preferred embodiment, activation of the signal generation means is triggered by a sequence of more than one event. In particular, the interaction between the analyte and the element (sensing element) suitably depends upon a separate xe2x80x9carmingxe2x80x9d event or reaction before the interaction can take place. For example, where the interaction involves a carrier or permeabilisation peptide such as alamethicin or GALA (SEQ ID NO:2), the xe2x80x9carmingxe2x80x9d event or reaction may comprise the adjustment of the pH of the assay medium in order to ensure that the peptide is active. The GALA (SEQ ID NO:2) peptide (WEAALAEALAEALAEHLAEALAEALEALAA (SEQ ID NO:3)) demonstrates insignificant triggering at neutral/alkaline pHs and triggering at lower pHs. The alternative KALA (SEQ ID NO:4) peptide (WEAKLAKALAKALAKHLAKALAKALKACEA (SEQ ID NO:5)) shows the reverse of no triggering at acidic pHs and triggering at neutral/alkaline pHs. This activity is retained even when a hapten is attached to the N terminus of the peptide.
Alternative methods of arming may be achieved by photoactivation of the peptides by conventional methods. For instance, a photoinduced conformational change can be introduced into the peptide structure (e.g. cis to trans conformation of stilbenes) to render the peptides active, or the photocleavage of a masking group.
The use of a separate arming event or reaction ensures that the level of non-specific reactions is minimised and therefore the level of background signal, from reactions not involving the analyte is reduced. This allows increased levels of sensitivity of the assay since although, in general, a signal as a result of an analyte would be expected to increase at a greater rate than that of the background signal, the level of the background signal can be the limiting factor in determining the lower limits of sensitivity of an assay.
In one embodiment, triggering of catalytic activity inside the nanospheres (FIG. 3) can be achieved according to this invention. The use of peptides specifically to reactivate inactive enzyme, to insert substrate or catalytic activity into a containment means has been investigated and is reported hereinafter.
Suitably the containment means are concentrated or separated from the assay medium before the signal is detected. For example, containment means may be concentrated by techniques such as sedimentation or aggregation using a suitable aggregating protein such as avidin. Centrifugation may also be employed where the containment means are of a suitable size.
Signals generated in accordance with the method of the invention may be detected using a variety of techniques. These include physical means such as optical detection methods (e.g. methods which detect absorption, fluorescence, fluorescence polarisation, time resolved fluorescence, chemiluminescence, bioluminescence, refractive index, evanescent waves, surface plasmon resonance, resonant mirror, Raman, light scattering, photoacoustic or photothermal spectroscopies), electrochemical detections methods (e.g. amperometric, potentiometric, conductimetric or dielectric detection methods) or electromechanical methods (e.g. gravimetric, surface acoustic wave, Love plate, or acoustic wave propagation methods). Of these, surface plasmon resonance detection may be particularly suitable.
Other techniques which may be used are those which detect individual particles by single particle detection methods,(e.g. Coulter Counting, flow cytometry) or by scanning microscopies (e.g. confocal scanning microscopy, scanning near field optical microscopy, scanning tunnelling microscopy, atomic force microscopy, scanning electrochemical and acoustic microscopies). Of these, flow cytometry may be particularly useful in the context of the present invention.
The fact that the signal is generated within a containment means is conducive to the use of single particle detection methods and in particular microscopic detection methods such as Coulter counters, flow cytometers, scanning microscopes or other sensors of similar dimensions. At present, the detection of many conventional fluorophores (e.g. Calcein, Fluorescein and Rhodamine) using these single particle detectors has proved difficult because the fluroescence of these dyes self-quench at desirable concentrations for detections. Using the method of the invention, these problems may be overcome. The accumulation of highly fluorescent deposits of the ELF-97 product in particular but also other excimer and exiplex fluorescent dyes in the containment means of the invention means that detection is now possible.
It is possible to detect and/or count single entities (i.e. a containment means in which the signal generating reagents have been activated). Accurate measurements of from 102 to 104 such entities can be readily achieved, although measurement of from 1 to 102 entities can be effected. As can be seen, this can equate to the presence of only a very few analyte molecules for example from 1 to 102 where the entity requires only a interaction with a single analyte to activate the signal generating means.
Single particle detection has been carried out as illustrated hereinafter using a modified Coulter Epics 5 cell sorter operating as a flow cytometer. This instrument was designed for operation on cells of size 1-100 microns. An instrument produced for operation in the 50-250 nm range is preferred in that it will allow lower noise and higher sensitivity allowing better discriminations between the water, background and test sample distributions as detailed in examples 24-26 below.
The Epics system is based on analogue detection using photon multiplier tubes (PMTs) with variable gain voltage and followed by post detection amplification. PMT""s operate with optimum signal to noise at a single gain voltage. In the Epics system the discriminator followed the amplifiers. The Epics system operated using analogue signals, but higher signal to noise may be achieved using photon counting detectors. It is also preferred that where the PMT""s are photon counting and the gain voltage is fixed, the comparater is placed immediately following the PMT""s and has a set value.
The Epics system uses a jet of fluid through air. This may cause significant background noise through scatter but the effect may be reduced by the use of a square walled flow cell. The analogue to digital converters (A/D) of the Epics system operated from a voltage of zero, where a very high gain is used to increase the distribution spacing the initial channels of the A/D are unused and the sensitivity of the instrument is decreased. A higher signal to noise may be achieved if the zero channel of the A/D may be set to equal the signal generated by a specific sample thus allowing a large increase in the number of A/D channels between size distributions.
More recent circuits than those of the Epics system may also operate with a higher signal to noise, higher bandwidth and reduced frequency response thus the instrument could be produced giving a higher signal to noise and increased distribution spacing.
As used herein, all measurements were taken using distilled double 0.22 micron filtered water, it is known in the art that higher grade filtering is available. All liposome suspensions were produced in standard buffers, it is known in the art that any of these contain significant background fluorescence and specialised buffers may be produced with a lower fluorescence background. Buffers and sheaf fluid may be degassed, or gassed with a specific gas, so to reduce background fluorescence. Buffers and sheaf fluid may or may not contain any of the additives which are known in the art to reduce background fluorescence.
Although the standard Epics system collects only a small percentage of the emitted light, the use of different lens combinations, mirrors and specialised flow cells may allow more light to be collected from the sample increasing the signal to noise ratio. Where the instrument uses a single laser line and that line pumps fluorescence, the scatter signal will be minimal as absorption of the light has occurred. The maximum fluorescence signal from a sample is fixed whereas the scatter signal may be increased, by increase of optical power.
An instrument may be produced using two or more excitation wavelengths. The first excitation wavelength designed to pump the fluorescent product, the second excitation wavelength to be outside the window of both the fluorescence and excitation and emission and used to produce a scatter signal. Preferably the scatter excitation will be a wavelength that is shorter than the excitation window thus increasing scattering.
Suitably the intensity of the fluorescence excitation beam will be of a magnitude just below that which would cause maximum fluorescence excitation such that scatter from impurities is reduced. Preferably the scatter excitation beam will be variable such that the excitation may be increased to clearly see the sample distributions whilst not being as high as to increase noise due to impurity scattering.
Very high laser powers may be used and these may be achieved using pulsed laser operation including, but not exclusive too Q-switching, modelocking and cavity dumping. Repetition rates of 100s of Mhz may be achieved and that laser pulses may be of the order of 100 femto seconds.
Time correlated single photon counting techniques can be used to further increase signal to noise of fluorescent measurement. Preferably the photon count from the both the fluorescence and scatter detectors will each be discriminated against two discrimination levels such that signals that are either two small or two large, to have been produced by the active species/particle, are ignored. Preferably the detectors will work as a logical AND combination reducing noise further. Preferably the fluorophore is chosen such that Raman scattering of the excitation beam by from the sample does not generate a signal in the emission area of the fluorophore.
Furthermore, such methods of the invention may be used in order to detect several analytes simultaneously. By providing a mixture of containment means such as liposomes which contain different signal generating agents and which are triggered by the presence of different analytes, the presence of more than one analyte may be detected.
It is an important advantage of some embodiments of the invention that a reaction of only one or a very few analyte molecules can give rise to a visible signal which may be counted indvidually using the techniques outlined above. The reason for this is that the signal generating agents are concentrated within a small particle, and not diffused throughout a sample volume. Furthermore, a definate signal, (the particles are either triggered or not) may result from the presence of one or very few analyte molecules.
This means that the levels of detection achievable with the method of the present invention is lower than with previously available methods. In order to achieve such high levels of sensitivity, the analysis of a large number of containment means (e.g. 106 or more) can be undertaken to ensure that those few which have been activated are detected.
Such highly sensitive detection methods can be used for example in detecting trace amounts of substances such as chemical or biological agents in samples or even in the air. Examples of chemicals which may be detected in this way include explosives, microorganisms and their products such as toxins, chemical warfare agents, pesticides, hormones or drugs.
Some embodiments of the invention may be considered to be more tolerant of variable levels of background signal, particuarly where the level of specific signal above background is only used to discriminate between counting of reacted versus non-reacted entities. In practice, it is possible, using the method of the invention, to combine counting of reacted entities and analysis of the quantity of signal in each reacted particle as an assay means. The quantity of signal in the reacted entities may be used to discriminate between specific reaction and non-specific reactions. Non-specific reacted entities may be distinguished by their different characteristics (e.g. optical) and/or simply the quantity of the signal detected in each entity. Non-specifically reacted entities may thereby be discarded from the counted signal. Accordingly, the counted signal will be that above a predetermined threshold or within a specific characteristic (e.g. optical) in each reacted entity analysed.
A containment means for use in a method as described above form a further aspect of the invention.
Yet a further aspect of the invention comprises a kit for detecting the presence of an analyte, said kit comprising a containment means enclosing signal generating reagents and an element which interacts specifically with said analyte so as to allow the activation of the signal generating reagents within the containment means.
As has been mentioned above, and is exemplified below, the applicants have found that the fluorescent output of a product is increased, and the output wavelength varied, by incubation of the substrate with a range of materials including cobalt ions as would not be foreseen in the art. In addition the relative quantities of conventional and incubated product that is used can affect the rate of the product reaction, which in itself, provides a signalling mechanism, particularly in the context of the process described above.
Also the incubation effect has been shown to increase the rate of product formation and modify the substrate in such a manner as to remain a substrate to an enzyme at pH ranges where the enzyme would not normally be active with the substrate.
That a fluorescent material may have its energy level increased by the addition of cobalt ions is quite unexpected in the art. Furthermore, it is surprising that a fluorescent material may have its energy levels shifted, thus causing an increase in the emitted intensity, by the addition of cobalt ions.
It was not foreseen that the fluorescent output intensity and/or wavelength of a product would be increased by means of incubation of materials such as metal ions with the substrate prior to product formation. It is surprising that the variation as described above is not reversible in that the variation only occurs substantially if the materials are added to the substrate and incubated. The shift does not occur if the same materials are added to the formed product that has been given sufficient time to stabilise.
The ELF reaction was found not to be stable immediately it reached its maximum fluorescent intensity but required up to ten hours to become irreversible in terms of the xe2x80x9cincubation reactionxe2x80x9d. The incubation effect may increase the rate of product formation. Furthermore, it may be employed in order to allow enzymes to operate outside the pH range that is possible with un-incubated substrate.
Where the signal (including but not exclusive to fluorescence) has a characteristic lifetime, the incubation effect will probably affect the lifetime of the signal.
All of these features give rise to potential assay applications.
Thus in a further aspect, the invention provides a method for modifying the signal from a signal generating reagent which generates said signal on reaction with a chemical activator, said method comprising incubating said signal reagent prior to exposure to a chemical activator, either with an ion, a surfactant and/or in a buffer at a p.H. which results in modification of a property of said signal.
Examples of such properties which may be modified include the wavelength of a signal emitted or the speed of the product formation, although other properties such as density, refractive index, and impedance may also be affected and these variations may also be measurable, for example using bulk sample analysis or single particle detection.
The signal generating reagent is suitably a dye, in particular a fluorescent dye such as ELF, an ELF substrate or a derivative or modified form thereof.
As well as cobalt ions, suitable modifying chemicals may be surfactants, in particular detergents such as Triton-X which may form a component of a buffer which are provided at an appropriate pH value.
The incubation effect can be utilised in the detection of modifying chemicals. Thus, a further aspect of the invention provides a method for detecting the presence of a chemical which modifies the signal from a signal generating reagent said method comprising contacting said signal generating reagent with a sample suspected of containing said chemical and subsequently with a chemical activator, and detecting the signal therefrom.
The incubation effects noted suggest that there may be binding of the modifying chemical to the substrate which produces a reduction in self-quenching. Such variation may cause a change in other measurable parameters such as density, refractive index and impedance and these variations may be measured by other suitable means that are known in the art in both bulk measurement and single particle detection
Many of the assay reactions show a significant time dependence over periods of hours (i.e. the reaction rate varies with time) and this is generally dependent on the level of analyte. Thus the measurement of the assay value or rate is preferably carried out at a predetermined time from the start of the reaction. A background signal measurement sample is suitably also measured simultaneously.
The effect of incubation time of the substrate and the irreversible nature of the reaction allows the dependence to be used as intrinsic measure of the rate of the reaction. This allows measurements to be made at any time during the reaction, and the results may be calibrated appropriately.
The amount of wavelength shift being an intrinsic measure of the reaction rates. It is further claimed that since this effectively integrates the rate over the entire experiment that it is possible to obtain a higher sensitivity at this point.
The invention is ideally suited to assays but may be used simply to produce novel markers. The term xe2x80x9cincubationxe2x80x9d is herein used to describe the process of contacting a substrate and a treatment additive for an appropriate period of time. This time period may vary between no preincubation as such and incubation over several days depending upon the precise nature of the reagents involved and the modifying effect required.